Most clinical chemistry assays—used both in human and veterinary clinical chemistry testing—measure ions, low molecule weight substances, enzymes, proteins etc. Most of these tests have been developed for human sample materials, but are also used to assay samples in veterinary medicine. Ions like sodium ions, potassium ions, manganese ions and chloride ions have identical structures in all species, and the same assay reagents may be used for determination of their concentration in samples of body liquids, cells and tissues from all species. This is also the case with small molecule substances like glucose, urea and creatinine, and most of the drugs administered to human or non-human mammals.
Enzymes from different mammalian species may have different structure, but most often they catalyse reactions of small molecule (substrates) which have identical structure in different mammalian species, and therefore the same reagents may be used to measure the enzyme activity of a class of enzymes in different mammalian species. For instance the enzyme lactate dehydrogenase may have different structure in different mammalian species, but catalyses the metabolism of lactate which has the same structure in all mammals, and the same reagents may therefore be used to measure lactate dehydrogenase enzyme's activities in samples from all mammals.
Larger molecules like proteins of different mammalian species most often differ in structure, although having the same biological function in said different species. Their concentration in samples of body liquids, cells or tissues of a species may be determined by the use of immunoassay methods, using antibodies raised in a different species by immunizing said different species with the antigen to be measured. By way of example, the concentration of human transferrin in human blood samples is often measured by using antibodies against human transferrin. These antibodies are usually made by immunizing another mammalian species (like goats, sheep, rabbits, rats or mice) with human transferrin, who recognises the human transferrin molecules as “foreign” and therefore produces antibodies towards human transferrin. There is an extensive literature on such antibody formation and immunoassay methods, e.g. “The Immunoassay Handbook” third edition by David Wild, Elsevier, Amsterdam, The Netherlands, ISBN 0 08 044 5268.
All immunoassays require standardisation. To obtain standardisation, a reference material is needed, which is used as the so-called “primary calibrator” for immunoassay measurements of an analyte. This is well-described practice (see chapter 9 of “Standardisation and Calibration” in “The Immunoassay Handbook”). Typically, the primary calibrators for protein immunoassays are based on the determination of a dry mass of a pure protein, as described by Søren Blirup Jensen in “Protein Standardization II: Dry Mass Determination procedure for the Determination of the Dry Mass of a Pure Protein Preparation” in Clin. Chem. Lab. Med. 2001; 39 (11): 1090-1097. Primary calibrators (also designated reference materials) can be made according to Blirup Jensen's protocol, or be purchased from specialised institutes like from the Institute for Reference Materials and Measurements, European Commission, Joint Research Centre. These specialised institutes provide ampoules of reference material for primary calibration of measurement systems.
“Secondary calibrators”, often also called “secondary standards” are often prepared in practice in view of the very limited availability of said primary standards or calibrators. In said “Immunoassay Handbook” the preparation of secondary calibrators (or secondary standards) from a target material is described. For this purpose, a calibration curve of the signals obtained from an immunoassay system using several dilutions of the primary calibrator is prepared, and then the secondary calibrators are measured. From these results an analyte concentration value is assigned to the secondary calibrator. A detailed protocol for development of secondary calibrators is found in Blirup Jensen et al. “Protein Standardization IV: Value Transfer Procedure for Assignment of Serum Protein Values from a Reference Preparation to a Target Material”, Clin. Chem. Lab. Med. 2001; 39 (11):1110-1122.
As described above, primary calibrators are used to assign analytical values to secondary calibrator materials. Immunoassay reagent manufacturers often make a secondary calibrator (with values assigned by using a primary calibrator), and store said secondary calibrator for internal use, and then use said secondary calibrator to assign values to routine calibrators which they sell to their customers. The latter calibrators could then be called a third generation calibrator, which could then be used to assign values to a fourth generation of calibrators, etc. Although proteins like albumin, transferrin, haptoglobin, haemoglobin and C-reactive protein (CRP) typically show structural and functional similarities between the different mammalian species they may be distinguished on the protein level. For example, human CRP differs from rabbit CRP on the protein level although there are many other similarities as well. Therefore, if a rabbit is immunised with human CRP, it forms antibodies towards human CRP. Dako AS, Denmark, and numerous other companies all over the world are selling such rabbit anti human CRP antibodies. However, these antibodies are not reactive to rabbit CRP (because the antibodies have been raised in rabbits), and cannot be used for immunoassays of rabbit CRP. Dependant on the actual degree of immunological similarity between the different species, said rabbit anti-human CRP antibodies may be used to measure dog CRP or goat CRP but the reaction is unpredictable, and probably significantly weaker, since all said species are mammals.
For example, rabbit anti human CRP antibodies (Dako product number Q 0329) are stated to be used for samples of human serum and plasma, samples from no other mammalian species are, however, suggested.
Other companies sell goat or sheep anti human CRP antibodies, raised in goat or sheep. These reagents cannot be used to measure goat or sheep CRP, respectively, and whether or to what extent they may be used in other mammalian species, is unpredictable. Most immunoassays used in veterinary medicine are therefore species-specific.
Dog CRP turbidimetric assays as those manufactured by TRID-Delta Company, Dublin, Ireland may be used for dog samples only.
Since many antigens have close structural relationships between different mammalian species, antibodies raised in one mammalian species against the antigen isolated from a second mammalian species may also cross-react with the same antigen from a third mammalian species. In the article “Evaluation of a commercially available human serum amyloid A (SAA) turbidimetric immunoassay for determination of equine SAA concentrations” by S. Jacobsen et al. in The Veterinary Journal 172 (2006) 315-319, the authors demonstrate that a commercial assay used (based on rabbit antibodies towards human SAA) could be used to determine SAA concentrations in body liquid samples from horses. However, no real assay was described, merely relative responses of different horse SAA were measured. Moreover, the system was calibrated with human SAA calibrator solutions.
Jonkisz et al report in Acta Veterinaria Hungarica (2010) 58(1), 59-67 on a comparison of turbidimetric and nephelometric assay results for Cystatin C in dogs. Both assay formats (PETIA and PENIA) were based on the use of rabbit anti-human Cystatin C antibodies, i.e. both assays were calibrated with human Cystatin C. No correlation between said two assay methods if applied to dog Cystatin C was observed, as illustrated by a correlation coefficient of merely 0.706 and it is suggested to consider carefully future validation procedures of PETIA and PENIA in the dog. Actually said results indicate that assays calibrated for an analyte of a different species are unreliable, most probably due to the completely unpredictable levels of cross-reactivity of the human anti Cystatin C antibodies with dog Cystatin C. Nevertheless, the authors of said paper did not consider an approach based on a calibration of these assay with dog Cystatin C.
In “The Veterinary Record” Sep. 13, 2008, 163, 327-330, Jacobsen et al. described in their article “Evaluation of a commercially available apparatus for measuring the acute phase protein Serum Amyloid A in horses” another immunoassay system where rabbit antibodies towards human SAA were used to determine the SAA concentration in body liquid samples from horses. In FIGS. 3 and 4 of said article, the authors point to the differences in calibration between the two systems and do not point to any attempt to validate the reported horse SAA protein concentrations in the samples analysed.
In Clinical Chemistry 49, No. 5, 2003, p. 810-813, Tsen et al. report in the article “Evaluation and Validation of a duck IgY Antibody based Immunoassay for High-Sensitivity C-reactive Protein: Avian Antibody Application in Clinical Diagnostics” on a duck IgY turbidimetric CRP measurement product from Good Biotech Corporation in Taiwan. This is the only known turbidimetric CRP assay using avian non-mammalian antibodies. At the end of said report the advantages of the use of avian antibodies in diagnostic human medicine are discussed. There is no hint whatsoever that these reagents have any universal use among all mammalian species.
In the article “Comparison between Chicken and Rabbit Antibody Based Particle Enhanced Cystatin C Reagents for Immunoturbidimetry” in Journal of Immunoassay & Immunochemistry, 29: 1-9, 2008, L.-O. Hansson et al. report on the advantages of the use of avian IgY antibodies (compared to mammalian antibodies) in particle enhanced immunoturbidimetric methods, which again only discuss the advantages for measurements of human samples. There is no hint to the advantage of using such antibody-based particles across the species boundaries among mammals.
Gassman et al. report in Faseb J. 4: 2528, 2532, 1990, the efficient production of chicken egg yolk antibodies against a conserved mammalian protein.
In Proc. Indian Natn Sci Acad. B69 No. 4 pp 461-468 (2003), in the article “Chicken Antibodies—Superior Alternative for Conventional Immunoglobulins”, Hodek and Stiborova mention that avian antibodies react with many epitopes due to the genetic differences between avians and mammals, and cause less interference in studies of human samples. Avian antibodies against rat CYP1A1 (a P450 enzyme) also react with human CYP1A1 at a very low detection limit. Hodek and Stiborova, however, do not raise the issue of calibration of the immunoassay response when cross-reactivity occurs. They also teach away from using turbidimetric methods using IgY, since they state that “the only limitation of chicken antibody application consists in the lower ability of IgY to precipitate antigens.” They do not propose the use of particle enhancement of the signal; instead they propose “however, using optimized reaction conditions, formation of precipitate can be facilitated (e.g. by using a higher ionic strength)”. Moreover, there is no mentioning of using calibrators with antigens from another mammalian species. They use preparations of human CYP1A1 when they test for human CYP1A1, and rat CYP1A1 when they test for rat CYP1A1.
In general, polyclonal antibodies are preferred over monoclonal antibodies in turbidimetric assays, since polyclonal antibodies solutions generally react with more epitopes on the antigens to be detected. The article “An automated turbidimetric immunoassay for plasma proteins” Bergström and Lefvert in Scandinavian Journal of Clinical and Laboratory Investigation, Vol. 40, 7, November 1980, 637-640, is one of many examples on how polyclonal antibodies are preferred over monoclonals in turbidimetric measurements. However, cocktails of monoclonals—or simply one monoclonal if the antigen is a polymer or consists of more than one subunit with the same epitopes—may be used, like in the Roche Diagnostics Tinaquant CRP assay.
In Immunology, 1992, 76(2): 324-330, Ying et al. report in the article “Reactivity of anti-human C-reactive protein (CRP) and serum amyloid P component (SAP) monoclonal antibodies with limulin and pentraxins of other species” that monoclonals to human serum proteins like CRP and Serum Amyloid A also may react towards said proteins from other mammalian species. The article indicates that this may occur or not, and cannot be generally relied upon. It is totally dependant on whether the epitope on the antigen—against which the monoclonal antibody is directed—is present in an unaltered structure in the different mammalian species.
Avian monoclonal antibodies exist, however, they have not come into use yet. Examples on such literature may be found in J. Biotechnol. 2006 Jul. 13; 124(2):446-56, with the title “Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments”, and in U.S. Pat. No. 6,143,559—“Methods for the production of chicken monoclonal antibodies” by Michael et al.